Studies of neuronally-derived nitric oxide (NO) have revealed many roles for this gaseous messenger molecule (Moncada, 1994; Yun et al., 1996). In the peripheral nervous system, NO mediates nonadrenergic, noncholinergic neurotransmission, serving as an effector of autonomic neurons on smooth muscle. NO has been implicated in several forms of neuronal plasticity, such as LTP (for a review see Huang, 1997). Studies in mice with a targeted genomic deletion of the NO biosynthetic enzyme, neuronal NO synthase (nNOS), have shown that NO mediates a substantial portion of the neurotoxicity associated with stroke (Huang et al., 1994). In the brain, NO and citrulline are produced from arginine predominantly by a neuronal isoform of NOS (nNOS) (Huang et al., 1993), although endothelial NOS (eNOS) may also occur in neurons (Dinerman et al., 1994; O'Dell et al., 1994). Most neurotransmitters are stored in synaptic vesicles and neurotransmitter effects are elicited following the exocytosis of transmitter into the synaptic space. For an evanescent transmitter such as NO there is no storage pool and newly synthesized NO is used as it is made. NO synthesis is triggered by the influx of calcium, which, when complexed with calmodulin, activates the biosynthetic activity of NOS (Bredt and Snyder, 1990).
Because NO lacks vesicular storage and depends on new synthesis for its release, nNOS must be associated with the plasma membrane. Subcellular fractionation indicates that roughly half of brain nNOS is soluble and half particulate (Bredt, 1996; Hecker et al., 1994). Recently, Bredt and associates showed that nNOS is targeted to membranes by binding to syntrophin, PSD95/SAP90, or PSD93 (Brenman et al., 1996; Brenman et al., 1996). These proteins are enriched in synaptic densities and interact with nNOS through PDZ domains, consensus sequences of about 100 amino acids that are found in proteins which tend to be associated with cell-cell junctions (Ponting and Phillips, 1995). The nNOS/PSD95 interaction involves a portion of nNOS which includes its sole PDZ domain and the second PDZ domain of PSD95. PSD95 was first isolated from postsynaptic densities (Cho et al., 1992) but also occurs in presynaptic nerve terminals (Kistner et al., 1993) and clusters neurotransmitter receptors and ion channels at synaptic sites (Kornau et al., 1997). For instance, the NMDA receptor and several potassium channels are associated with PSD95 at synapses (Kornau et al., 1995). The linking of NMDA receptors to nNOS by PSD95 may explain why calcium influx following NMDA receptor activation leads to a tightly coupled nNOS activation (Brenman et al., 1996). Indeed, the effects of NO appear to be intimately tied to the NMDA receptor. For example, NMDA receptor-mediated neurotoxicity (Dawson and Dawson, 1996), neurotransmitter release (Schuman and Madison, 1994), and cGMP elevations (Bredt and Snyder, 1989; Garthwaite et al., 1989) each require nNOS and are blocked by nNOS-specific inhibitors. Moreover, NO can directly modulate NMDA receptors (Lipton and Stamler, 1994).
There is a continuing need in the art of neurotransmitter regulation for methods of affecting the activity of neuronal NOS, so that one can manipulate NO levels when required for therapeutic effect in such disorders.